25 µl of ICAM-1/Fc at 50 µg/ml in 0.1 M NaHCO3 (pH 8.6) was adsorbed to the center of a 35 mm tissue culture dish (Falcon 353001, Becton Dickinson Labware, FranklinLakes, NJ) overnight at 4°C. We found that using NaHCO3 allows for the protein to adhere to the dish better. Unbound ICAM-1/Fc was removed by rinsing the dish threetimes with PBS. Experiments were done using RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Irvine Scientific, Santa Ana, CA), penicillin (50 U/ml, Gibco BRL, Grand Island, NY) and streptomycin (50 µg/ml, Gibco BRL). 2ml of the RPMI medium were added to the ICAM-1 coated dish for 30 minutesbefore the experiment and prior to the addition of 3A9 cells. The fetal calf serum in the medium was used to block the exposed surface of the dish.

(A) Photograph of our AFM set-up. The CCD camera is not in view. (B) Complete schematic diagram of the AFM.

Fig. 3

Steps in the acquisition of an AFM force measurement.

The first step is the approach of the cantilever with a cell bound to the substrate. This is followed by contact between the cell and substrate and retraction of the cantilever, which results in the separation of the cell from the substrate. The cantilever is bent during this process. The arrows indicate the direction of cantilever movement.

A piezoelectric translator was used to lower the cantilever/cell onto the sample. The interaction between the attached 3A9 cell and the sample was given by the deflection of the cantilever, which was measured by reflecting a laser beam off the cantilever into a position sensitive 2-segment photodiode detector. AFM cantilevers were purchased from TM Microscopes (Sunnyvale, CA).

In order to obtain measurements of unitary LFA-1/ICAM-1 unbinding forces, we used conditions that minimized contact between the 3A9 cell and the sample. An adhesion frequency of <30% in the force measurements ensured that there is a >85% probability that the adhesion event is mediated by a single LFA-1/ICAM-1 bond (3).

(A) Tip of the AFM cantilever indenting a 3A9 cell. The cell compliance measurements were based on the assumption that the cell is an isotropic elastic solid and the AFM tip is a rigid cone (20-22). According to this model, initially proposed by Love and Hertz, the force (F)-indentation (a) relation (shown) is a function of Young’s modulus of the cell, K, and the angle formed by the indenter and the plane of the surface, θ, as follows: F= [(K/2(1-v2)][4/π tan θ][α2]. We assumed the indenter angle, θ, formed by the AFM tip and the 3A9 cell to be 55° and Poisson ratio, n, to be 0.5. (B) Schematic of an AFM cell compliance measurement. During the approach, the cantilever is lowered onto the center of a 3A9 cell. Then the cell is indented with the tip of the cantilever. Arrows indicate direction of cantilever movement. (C) Force versus indentation traces of resting, PMA-stimulated and Mg2+-treated 3A9 cells. (D) Young’s modulus of resting, PMA-stimulated and Mg2+-treated 3A9 cells. The data is based on 15-30 elasticity measurements per cell done on 25 different 3A9 cells in each case. The error bar is the standard error.

Results and Discussion

An AFM force measurement of a receptor-ligand interaction between a cell and an opposing substrate coated with ICAM-1 involvesfourmainsteps that are shown in Figure 3. First, the cantilever with the 3A9 cell attached is lowered onto an ICAM-1-coated dish. Contact is made, allowing for the receptor-ligand interaction to take place. Then, the cantilever is retracted via the contraction of the piezoelectric translator, pulling the LFA-1/ICAM-1 bondsapart. Finally, complete separation of the two is achieved, and the process can be repeatedagain. During both the approach and retraction events, the cantilever is bent and the tension between the LFA-1 on the cell and ICAM-1 is determined from the deflection of the cantilever.

(A) Multiple-bond measurements acquired with a compression force of 200 pN, 5 seconds contact and a cantilever retraction speed of 2µm/second. The measurements were carried out with a resting cell (1st trace), a Mg2+-treated cell (2nd trace), and a PMA-stimulated cell (3rd trace). The 4th trace corresponds to a measurement acquired from a PMA-stimulated cell in the presence of LFA-1 (20 µg/ml FD441.8) and ICAM-1 (20 µg/ml BE29G1) function-blocking monoclonal antibodies (mAbs). Arrows point to breakage of LFA-1/ICAM-1 bond(s). ƒde is the detachment force and the shaded area estimates the work of de-adhesion. (B) Single-molecule measurements of LFA-1/ICAM-1 unbinding forces. Traces 2 and 5 show adhesion. Measurements were obtained under conditions that minimized contact between the 3A9 cell and the ICAM-1 coated surface. The compression force was reduced to ~60 pN and the contact time to 50 miliseconds. An adhesion frequency of less than 30% in the force measurements ensured that there is a >85% probability that the adhesion event is mediated by a single LFA-1/ICAM-1 complex (18). The frequency of adhesion in test and control experiments was examined to confirm the specificity of the interaction (18, 19). The addition of monoclonal antibodies against either LFA-1 or ICAM-1 significantly lowered the frequency of adhesion of both resting and activated cells under identical experimental conditions. Both resting and stimulated 3A9 cells exhibited lower frequency of adhesion to immobilized bovine albumin than to immobilized ICAM-1.

The parameters mentioned above allow us to determine changes in adhesion. Both the compression force and contact time have an impact on adhesion. For example, a greater compression force and a longer time of contact will result in greater adhesion. Adhesion can also be modulated by cell activation. The force scan of a resting cell has fewer bonds formed and a smaller area of de-adhesion and detachment force than force scans of cells treated with agentspromoting adhesion. We used Mg2+, which has been found to activate LFA-1 (23, 24). The result of Mg2+ activation is an increase in the number of bonds formed between the two complexes and in the work of de-adhesion and detachment force (Fig. 4A) (2, 3). We also stimulated the cells using phorbol- myristate acetate (PMA). It activates a protein kinase C pathway that leads to enhanced adhesion (25-27). With this agent, we see an even greater increase in the number of bonds formed as well as a great increase in the work of de-adhesion and detachment force (Fig. 4A) (2, 3). It is essential to confirm the specificity of any interaction. For the LFA-1/ICAM-1 interaction, this can be done with readilyavailable antibodies for both LFA-1 and ICAM-1. The last force scan of Figure 4A represents an ICAM-1/LFA-1 interaction that had been blocked with an antibody. After blocking both the ligand and receptor, almost all adhesion is eliminated (2, 3).

The AFM allows us to examine single molecule interactions. There is a greater than 85% chance that a single molecule interaction is being measured if the adhesion frequency is reduced to less than 30%. This is achieved by reducing both the duration of contact between the cell and ICAM-1 as well as the applied compression force. In the ICAM-1/LFA-1 experiments contact was reduced to ~50ms and the compression force to ~60pN.

Single molecule measurements can be seen in Figure 4B. Adhesion onlytakes place in the second and fifth force scans, and it is a single molecule breakage. The unbinding force is calculated from the magnitude of the force transition with corrections for hydrodynamic drag. In order to determine the force versus loading rate profile, the force spectra were first plotted versus piezo displacement. Loading rates were obtained by multiplying the slope of the force versus displacement curve with the retraction speed of the cantilever. The resulting force versus loading rate relationship always showed an increase of unbinding force with increasing loading rate (2, 3).

The mechanical properties of the cell were determined through AFM indentation measurements of cell compliance. The indentation force used was below 1nN (~600 pN). In order to satisfy the constraints of the Hertz model, it is important to considerindentations of less than 10% of the diameter of the cell (28). The 3A9 cells used in our studies were between 10-15 µm in diameter. Therefore, we only considered indentations of less than 1µm.

In order to determine the cell’s elasticity, the force versus indentation measurements were fitted to the curves of the Hertz model (Fig. 5C). In our experiments it was important to determine the elasticity of the 3A9 cells in the different conditions that were used for activating the cells. We found that the cells treated with PMA had the lowest Young’s modulus values and were, therefore, the most compliant. This wouldlead to a greater degree of spreading by these cells during AFM force measurements and offered an explanation for the large work of de-adhesion that was observed with the force scans of the PMA- treated cells (Fig. 5D) (2).

Outside noise can severely interfere with obtaining data. In order to block out acoustical and mechanical noise, our set-ups are shielded inside of acoustic/vibration isolation chambers. This also aids in maintaining the temperature. For our experiments it is important to keep the temperature above 25ºC to keep the cells alive.

As described in the methods and protocol sections, cells were attached via biotin- BSA, streptavidin, and finally concanavalin A (conA) as is presented in Figure 2. The cells used in these types of AFM experiments must have surface receptors for conA for the described method of attachment. We use 3A9 and K562 cells in our experiments.

The strength of the conA attachment is many times stronger than the interaction of LFA-1 and ICAM-1 (2nN versus 50 pN). This is crucial for these types of measurements because if the receptor/ ligand interaction being studied is stronger than the conA linkage, then the cell will come off the tip. If this should occur one will only observe one measurement resulting from the conA linkage breaking. Normally, we are able to obtain hundreds of measurements on the LFA-1/ ICAM-1 system without the cell coming off the cantilever tip.

When attaching a cell to the cantilever, it is best to keep the concentration of cells in the tissue culture dish lowenough so that there are only a few cells in the field of vision. This makes it easier to determine whether a cell had attached to the cantilever. Also, a high cell concentration can increase the likelihood of an extra cell attaching to the cantilever tip, which could interfere with results. The extra cells can also stick to the cell that is already attached to the cantilever. In this case, the measurements would be complicated by cell to cell interactions that would be recorded in addition to the interaction of the cell with the protein substrate on the bottom of the dish.

During elasticity measurements, the cell surface is probed with the cantilever tip. In our experiments, we had used 3A9 cells that are 10 µm in diameter. Using considerably smaller cell types could be difficult. It is important to probe the center of the cell with the cantilever tip, as the cell can veryoftenslip out from under the cantilever. One must continuously make sure that the tip is stilltouching the cell and not the surface of the tissue culture dish. Probing the dish surface will result in extremely high Young’s modulus values, as the dish is much stiffer than a cell. To avoid this, it is best to turn off the laser every few measurements and turn on the light to clearlyinspect the position of the cantilever. Often, it may be helpful to do a few measurements with the light on in order to visualize the path of the cantilever and where it is touching the cell surface. If the tip is touching the cell considerably off-center, the cell is more likely to slip out from under the cantilever.

Learning how to operate the AFM can take more time than performing a simple adhesion assay for the first time. However, there are many benefits to adapting this technique. The AFM is a versatile tool that can be used to study cell adhesion as well as cell compliance. As we described, cell adhesion studies can be performed at both the multiple bond and the single molecule level providing a wealth of information about a particular receptor-ligand system. Cell compliance studies can complement the adhesion studies with information concerning the mechanical properties of the cell.

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References

Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1.

F Sanchez-Madrid et. al

J Exp Med, 1983

Contributions of molecular binding events and cellular compliance to the modulation of leukocyte adhesion.

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